Physiological & Behavioral Indicators of Shad Susceptibility to Impingement at Water Intakes

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1 University of Tennessee, Knoxville Trce: Tennessee Reserch nd Cretive Exchnge Msters Theses Grdute School Physiologicl & Behviorl Indictors of Shd Susceptibility to Impingement t Wter Intkes Brooks Ashley Fost University of Tennessee - Knoxville Recommended Cittion Fost, Brooks Ashley, "Physiologicl & Behviorl Indictors of Shd Susceptibility to Impingement t Wter Intkes. " Mster's Thesis, University of Tennessee, This Thesis is brought to you for free nd open ccess by the Grdute School t Trce: Tennessee Reserch nd Cretive Exchnge. It hs been ccepted for inclusion in Msters Theses by n uthorized dministrtor of Trce: Tennessee Reserch nd Cretive Exchnge. For more informtion, plese contct trce@utk.edu.

2 To the Grdute Council: I m submitting herewith thesis written by Brooks Ashley Fost entitled "Physiologicl & Behviorl Indictors of Shd Susceptibility to Impingement t Wter Intkes." I hve exmined the finl electronic copy of this thesis for form nd content nd recommend tht it be ccepted in prtil fulfillment of the requirements for the degree of Mster of Science, with mjor in Wildlife nd Fisheries Science. We hve red this thesis nd recommend its cceptnce: Mrk S. Bevelhimer, Chrles C. Coutnt, Mrshll S. Adms (Originl signtures re on file with officil student records.) Richrd J. Strnge, Mjor Professor Accepted for the Council: Dixie L. Thompson Vice Provost nd Den of the Grdute School

3 To the Grdute Council: I m submitting herewith thesis written by Brooks Ashley Fost entitled Physiologicl & Behviorl Indictors of Shd Susceptibility to Impingement t Wter Intkes. I hve exmined the finl electronic copy of this thesis for form nd content nd recommend tht it be ccepted in prtil fulfillment of the requirements for the degree of Mster of Science, with mjor in Wildlife nd Fisheries Science. Richrd J. Strnge Mjor Professor We hve red this thesis nd recommend its cceptnce: Mrk S. Bevelhimer Chrles C. Coutnt Mrshll S. Adms Accepted for the Council: Anne Myhew Vice Chncellor nd Den of Grdute Studies (Originl signtures re on file with officil student records.)

4 Physiologicl & Behviorl Indictors of Shd Susceptibility to Impingement t Wter Intkes A Thesis Presented for the Mster of Science Degree The University of Tennessee, Knoxville Brooks Ashley Fost My 2006

5 Acknowledgements I wish to thnk ll those who helped me complete my Mster of Science degree in Fisheries Science. I would like to thnk Dr. Mrk Bevelhimer for his tireless guidnce nd effort throughout the lst yer. I would lso like to thnk Dr. Chuck Coutnt nd Dr. Mrshll Adms for ll of their ssistnce nd guidnce in my reserch. The experience nd knowledge gined from these mentors ws priceless. I would lso like to thnk Dr. Richrd Strnge for giving me the opportunity to obtin this degree nd for ll of his encourgement. Thnks to Ok Ridge Ntionl Lbortory for fcilities use nd the Electricl Power Reserch Institute for reserch funding tht mde this possible. Lstly, I would like to thnk my fincé, fmily, nd friends who supported me dily nd whose encourgement mde the entire process so much esier. ii

6 Abstrct To ddress Clen Wter Act regultions concerning reduction of fish impingement t wter intkes (316b) I investigted pproches for identifying moribund or impired shd under stressors of cold shock nd reduced rtion. In preliminry experiments, we determined the initil point of loss of equilibrium (LOE) for thredfin shd to be 7.2 C nd for gizzrd shd to be less thn 3.5 C. I exposed shd to incresing levels of cold shock nd reduced rtion nd then mesured physiologicl nd behviorl responses. Gizzrd nd thredfin shd exhibited reduced swimming performnce t tempertures slightly bove LOE. Cortisol nd chloride showed liner correltion with swimming performnce in gizzrd shd but not thredfin shd. Cortisol incresed s response to cold shock nd declined fter n cclimtion period t cold temperture. The expected chnge in serum chloride in reltion to cortisol occurred in thredfin shd but not gizzrd shd. I observed few differences in swimming performnce nd physiologicl indictors mong rtion tretments for either species. However, field observtions suggest tht the durtion of reduced rtion I used, 21-dys, ws not sufficient to reproduce the condition of shd in lte winter. The nutritionl sttus indictors, hemtocrit nd condition fctor, demonstrted declining trend. These indictors could be mesured esily in the field to determine the susceptibility of shd if further testing shows reltionship with decresed swimming performnce. The results of this study indicte tht the term moribund does not pply well to ll shd impcted by nturl environmentl stressors nd its ppliction should be reevluted. Thredfin nd gizzrd shd re susceptible to impingement before moribundity occurs. The use of physiologicl indictors s predictors of impingement susceptibility ppers promising. iii

7 Tble of Contents I. INTRODUCTION...1 II. METHODS AND RESULTS...7 Generl Methods...7 Indictors of Cold Shock nd Effects on Swimming Performnce...13 Methods Results Gizzrd Shd Thredfin Shd Indictors of Combined Coldshock nd Reduced Rtion nd Effects on Swimming Performnce...22 Methods Results Gizzrd Shd Thredfin Shd III. DISCUSSION...27 Prcticl Implictions nd Future Studies...33 LIST OF REFERENCES...35 APPENDIX...39 VITA...45 iv

8 List of Figures Figure 1. - Thredfin shd (NtureServe 2005)....3 Figure 2. - The current (green) distribution of thredfin shd in the U.S. (NtureServe 2005)...3 Figure 3. - Gizzrd shd (NtureServe 2005)....4 Figure 4. - The current (green) distribution of gizzrd shd in the U.S. (NtureServe 2005)...4 Figure 5. - The gizzrd shd collection site, circled in red, ner Clinch River km 15 (U.S. Army Corps of Engineers 2004)....8 Figure 6. - The thredfin shd collection site, circled in red, t Clinch River km 23, Tennessee (U.S. Army Corps of Engineers 2004)....9 Figure 7. - Summry of the protocol used during the course of cold shock nd reduced rtion experiments for gizzrd nd thredfin shd. Procedures tht re specific to prticulr experiment or not performed on controls re lbeled in prentheses Figure 8. - Schemtic of the swimming performnce chnnel from the top down perspective. Fish were confined to the 10.8 cm wide by 122 cm long re during testing Figure 9. - Tretment grph displying the points (circles nd squres) where gizzrd nd thredfin shd were smpled during 0.5 C/hour cold shock experiments. Circles indicte groups smpled immeditely when the test temperture ws reched. Squres indicte groups smpled 3 or 6 hours fter reching the test temperture...14 Figure 10. Loss of equilibrium, s function of time nd temperture, for gizzrd nd thredfin shd subjected to cold shock tretment tht begn t 15 C nd declined t rte of 0.5 C/hour. The intended temperture decline is indicted by the line. Open dimonds re for shd tht lost equilibrium. Tringles re for shd tht were removed prior to LOE...16 Figure Men (+1 SE) swimming time of gizzrd shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Groups were tested t 15 C (control), 5 C, fter 6 hours t 5 C (5C + 6Hrs), 4 C, nd fter 6 hours t 4 C (4 C + 6 hours). Tretments tht re sttisticlly different (P<0.05) hve different letters Figure Men (+1 SE) condition fctor of gizzrd shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Tretments tht re sttisticlly different (P<0.05) hve different letters Figure Men (+1 SE) plsm cortisol nd chloride of gizzrd nd thredfin shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Tretments tht re sttisticlly different (P<0.05) hve different letters...18 v

9 Figure Liner correltion of men cortisol to men swim time of thredfin nd gizzrd shd...19 Figure Liner correltion of men chloride to men swim time of thredfin nd gizzrd shd...19 Figure Men (+1 SE) swimming time of thredfin shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Groups were tested t 15 C (control), 8.5 C, 8.5 C + 6 hours, 7.5 C, nd 7.5 C + 3 hours. Tretments tht re sttisticlly different (P<0.05) hve different letters...21 Figure Men (+1 SE) condition fctor of thredfin shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Tretments tht re sttisticlly different (P<0.05) hve different letters Figure Men (+1 SE) swimming time of gizzrd nd thredfin shd exposed to cold shock fter one of three protocols:14 dys of full rtion, 14 dys of reduced rtion, or 21 dys of reduced rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters...24 Figure Men (+1 SE) plsm cortisol nd chloride of gizzrd nd thredfin shd exposed to cold shock fter one of three rtion tretments:14 dys of full rtion, 14 dys of reduced rtion, or 21 dys of reduced rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters...24 Figure Men (+1 SE) condition fctor, hemtocrit, plsm totl protein, nd plsm triglycerides of gizzrd nd thredfin shd exposed to cold shock fter one of three rtion tretments:14 dys of full rtion, 14 dys of reduced rtion, or 21 dys of reduced rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters Figure A4. - Two hybrid shd t top of picture followed by three thredfin shd nd single gizzrd shd. Notice the similrity in fin colortion but different snout shpe. The hybrid lso hd the long dorsl fin filment (not visible in picture) chrcteristic of thredfin shd Figure A5. Gizzrd shd (top) compred to two hybrid shd (middle nd bottom). Notice the yellow fin colortion, rounded overhnging snout, nd long dorsl filment. 44 vi

10 I. Introduction In 2004, the United Sttes Environmentl Protection Agency (USEPA) published the Clen Wter Act 316(b) phse II rule pertining to existing power genertion fcilities. The phse II rule estblished performnce stndrds for cooling wter intke structures tht required lrge reduction in impingement mortlity reltive to the clcultion bseline. This bseline refers to the impingement mortlity tht would theoreticlly occur if the fcility hd shoreline, ner-surfce intke with stndrd 3/8- inch mesh trveling screen with its fce oriented prllel to the shoreline, but no other mesures to reduce impingement mortlity. The rule sttes tht reduction by 80 to 95 percent from the clcultion bseline must be chieved regrdless of the type of source wter body. Studies hve shown nturl mortlity of shd in lkes nd reservoirs in winter corresponds to pek impingement periods (Griffith nd Tomljnovich 1975; Lor et l. 1978; Griffith 1978; McLen et l. 1980, 1982; Adms et l. 1985; White et l. 1986). The USEPA hs recognized the need to identify nturlly moribund (dying) fish nd requires, s prt of verifiction monitoring pln tht impingement hs been reduced, proposl on how nturlly moribund fish tht enter the cooling wter intke structure will be identified nd quntified when determining if performnce stndrds of 80-95% reduction hve been met. Two fctors, cold shock nd reduced rtion, re relted to the nturl mortlity of shd. In the Southest US, over 90% of fish impinged on cooling-wter intke screens of therml power sttions re thredfin shd (Dorosom petenense) or gizzrd shd (D. cepedinum) (EPRI, 2005). Thredfin shd re bundnt forge fish found in lkes nd 1

11 reservoirs of the southest (Figures 1 nd 2). The species nturl rnge in gulf cost sttes hs been expnded through stocking by fisheries gencies, which sought smll, highly prolific prey species for gme fish such s lrgemouth bss (Micropterus slmoides). The popultions occurring in the species current northern rnge re subject to cold tempertures tht cn cuse loss of equilibrium (LOE) or deth. A rnge of tempertures cusing loss of equilibrium nd mortlity hve been reported for thredfin shd. Griffith (1978) found mortlity to occur t tempertures s high s 9 C nd 100% mortlity by 4 C. Impingement of thredfin shd increses significntly when wter tempertures drop below 7 C (McLen et l. 1985). Gizzrd shd re n bundnt forge fish tht rnge from the Midwest to the Est cost nd s fr north s the Gret Lkes region (Figures 3 nd 4). Gizzrd shd ccount for lrge percentge of impingement in the Gret Lkes region where tempertures often rech the reported LOE temperture for this species. A rnge of LOE tempertures hs been reported for gizzrd shd. Cox nd Coutnt (1976) showed the LOE to be lower thn 6.5 C. Neumnn et l. (1977) reported the species cn survive t tempertures below 1 C for short period. Heidinger (1983) suggested tht mortlity occurs in gizzrd shd t tempertures between 4 nd 0 C. Impingement likely increses when shd re subjected to tempertures tht ffect their physiologicl function nd performnce. Susceptibility to impingement occurs t some point bove the LOE temperture for both species nd is the prefce to nturl mortlity probbly induced by cold shock. The premise for this reserch is tht the mount of cute nd chronic stress prior to LOE cn be quntified using bioindictors of stress. The use of plsm cortisol nd chloride to quntify sub lethl stress responses in fish is well estblished (Brton et l. 2002, Strnge nd Schreck 1978). White et l. 2

12 Figure 1. - Thredfin shd (NtureServe 2005). Figure 2. - The current (green) distribution of thredfin shd in the U.S. (NtureServe 2005). 3

13 Figure 3. - Gizzrd shd (NtureServe 2005). Figure 4. - The current (green) distribution of gizzrd shd in the U.S. (NtureServe 2005). 4

14 (1986) used plsm cortisol nd chloride to quntify stress response to cold shock in gizzrd shd. Reduced rtion hs lso been shown to ffect the susceptibility to nturl mortlity in gizzrd shd (Adms et l.1985). The role of reduced rtion in thredfin shd nturl mortlity hs not been investigted, prticulrly in colder periods. Lipids re typiclly stored during periods of high food vilbility (summer nd fll) nd utilized during periods of low food vilbility or nonfeeding periods (winter nd erly spring) (Adms 1999). The influence of feeding t cold tempertures nd durtion of strvtion on susceptibility to impingement hs not been quntified. Bodol (1966) reported gizzrd shd to discontinue feeding t 11 C. Both species remin ctive but lethrgic during periods of cold tempertures, requiring the utiliztion of energy reserves to mintin physiologicl homeostsis. The physiologicl condition of fish, quntified using bioindictors or condition indices, could revel the role of rtion in nturl mortlity. Hemtocrit, triglycerides, nd totl protein hve been used s generl indictors of nutrition nd strvtion (Adms et l. 1985, 1992; Brton et l. 2002). Condition fctor (K), n index tht reltes weight nd length, hs been used s n integrtion of energy storge nd metbolism due to strvtion (Dutil et l. 2003). Swimming performnce is importnt for relting physiologicl condition to impingement. Griffith nd Tomljnovich (1975) used swimming performnce to determine the bility of cold shocked thredfin shd to void impingement nd found tempertures below 8 C cused high impingement mortlity. Mrtinez et l. (2004) demonstrted tht Atlntic cod (Gdus morhu) tht were strved hd reduced swimming performnce compred to cod tht were fed. However, the combined ffects of rtion nd cold shock on swimming performnce hs not been investigted. 5

15 The chllenge for power sttion opertors is to determine whether fish impinged on intke screens would hve died nywy becuse of environmentl conditions. A rpid nd simple procedure for ssying fish is desired, either using fish lredy impinged or those collected in the intke foreby. The primry purposes of this study were to: 1) identify the criticl points where cold shock nd reduced rtion ffect the bility of fish to escpe impingement nd 2) identify physiologicl & performnce indictors for identifying susceptibility of fish to impingement. I first determined the temperture tht cused ner-lethl response by subjecting shd to cold shock (strting t 15 C nd reduced by 0.5 C/Hour) nd observing individul LOE. Shd were then cold shocked using the sme decline rte over rnge of tempertures slightly bove the LOE temperture to quntify stress nd swimming performnce response. A lb study combining full nd reduced rtion groups pired with single cold shock regime to quntify stress nd swimming performnce response ws lso performed. Blood ws withdrwn from individuls to identify physiologicl indictors of stressed fish. Swimming performnce of individul fish ws ssessed using strem chnnel with velocity similr to tht deemed cceptble by USEPA for power plnt intke (~0.15 meters/sec). 6

16 II. Methods nd Results Generl Methods Gizzrd nd thredfin shd were collected by electrofishing from August to October 2005 on the Clinch River, Tennessee, United Sttes. Gizzrd shd were collected from single cove (ner Clinch River kilometer 15, Figure 5) nd thredfin shd from the dischrge re of the Tennessee Vlley Authority s Melton Hill Dm (Clinch River kilometer 23, Figure 6). Wter tempertures rnged from C. I trnsported live shd to Ok Ridge Ntionl Lbortory in 151 L brrels equipped with two bttery operted ertors nd treted with 400 g of sodium chloride. Shd were then held t 24 C for 3 to 5 dys in 889 L circulr tnks. The tnks mesured 122 cm in dimeter nd 76 cm in height. Ech tnk ws equipped with n ertor nd bout 0.6 L/min flow through. Shd were cclimted to feeding of frozen brine shrimp during this period. Test groups of shd were held in one of four 530 L rectngulr tnks receiving 0.25 L/min of flow. This llowed testing of 4 groups t time. Test groups of gizzrd shd numbered 34 individuls with men length of 153 mm ( mm) nd weight of 30 g (17-47g). Test groups of thredfin shd included 45 individuls with men length of 134 mm ( mm) nd weight of 30 g (9-30g). The number of individuls in ech test group exceeded the number required for testing to llow for holding mortlity. Portble refrigertion units pired with temperture controllers were used to regulte exposure tempertures within ± 0.2 C. Ech test group ws cclimted for one week t 15 ± 0.2 C prior to testing in one of two experiments. The protocol used for the two experiments is illustrted in Figure 7. A single test group for ech experiment ws 7

17 Figure 5. - The gizzrd shd collection site, circled in red, ner Clinch River km 15 (U.S. Army Corps of Engineers 2004). 8

18 Figure 6. - The thredfin shd collection site, circled in red, t Clinch River km 23, Tennessee (U.S. Army Corps of Engineers 2004). 9

19 Acclimte Test Group t 15 C for 7 dys (Cold Shock) Cold 0.5 C/hr to 4, 5, 7.5, or 8.5 C (except controls) (Reduced Rtion) Feed 0.5% rtion for 14 or 21 dys (except controls) Hold 0, 3, or 6 hours t Test Temperture (except controls) Cold 0.5 C/hr to 5 or 8.5 C Collect Blood Immeditely Swimming Performnce Test Collect Blood Immeditely Swimming Performnce Test Figure 7. - Summry of the protocol used during the course of cold shock nd reduced rtion experiments for gizzrd nd thredfin shd. Procedures tht re specific to prticulr experiment or not performed on controls re lbeled in prentheses. repeted for ech species. Repeted cold shock experiments were conducted 4 weeks fter the initil test group for gizzrd shd nd three weeks lter for thredfin shd. The repeted reduced rtion groups were conducted one dy lter thn the initil 21-dy test group. Eighteen gizzrd or 24 thredfin shd were removed from their exposure temperture prior to collection of blood for serum nlysis. I removed shd from the exposure tnks with smll dip nets tht llowed for quick removl of individuls to minimize hndling stress nd immeditely nesthetized in solution of tricine (MS- 222). Only three shd were removed t time nd bled within 2 to 4 minutes of being plced in MS-222. Fish were bled using 21G1 Vccutiner needles pired with 13 X 75 mm heprinized Kendll collection tubes. I mesured totl length (mm) nd weight (g) of ech individul. Blood smples were rndomly pooled, due to the low volume of serum derived from individuls, to form six pool groups of 3 gizzrd or 4 thredfin shd for ech tretment. Hemtocrit ws mesured for ech pooled smple. Plsm ws 10

20 seprted from whole blood by centrifugtion, trnsferred to 1.5-mL cryotubes, nd stored in liquid nitrogen for lter nlysis. Plsm cortisol concentrtion ws determined vi Cot-A-Count solid-phse 125 I-cortisol rdioimmunossy. Plsm chloride ws determined using spectrophotometric ssy by Pointe Scientific. For swimming performnce tests, groups of ten gizzrd or thredfin shd were removed from their tretment tnks (the sme tnks from which the fish to be bled were tken) nd plced into the corrl re of the swimming performnce chnnel (Figure 8). The dimensions of the test chnnel nd methods of the test llowed 5 fish to be tested simultneously. The swim chnnel ws mintined t the temperture fish were exposed to within their tretment using portble refrigertion units. The flow (~0.15 meters/sec) in the test chnnel ws produced by ¾ hp centrifugl pump. Wter ws pumped from the corrl zone nd introduced to the upper end of the test chnnel through series of incresing-dimeter pipes nd 0.32 cm mesh screen to even the flow distribution within the test zone of the chnnel. The performnce test chnnel ws 10.8 cm wide X 122 cm long. Wter depth ws held t 14.6 cm. The power to the pump ws surged 3-4 times to llow the fish to orient upstrem nd gin swimming blnce prior to inititing full flow velocity. Ech individul ws observed during mximum 1 hour period to determine if impingement occurred t the rer screen for > 15 sec. The totl swim time ( 1 hour), totl length, nd weight of ech individul ws recorded. Condition fctor (weight / (length^3) * 1000) ws clculted for individuls. Sttisticl nlyses on ll dt were performed using SAS, version 9.1, nd SPSS, version 14. A vlue of P<0.05 ws considered significnt for ll tests nd simultneous 11

21 Pump Performnce Test Chnnel Scle (m) Chiller Corrl Are 0 0.5m Figure 8. - Schemtic of the swimming performnce chnnel from the top down perspective. Fish were confined to the 10.8 cm wide by 122 cm long re during testing. 12

22 confidence ws held t P=0.05 for ll post hoc tests. Correltions between vribles were investigted using Person correltion coefficients. Differences between test groups in swimming performnce nd physiologicl indictors were nlyzed with nlysis of vrince (ANOVA). The Shpiro-Wilk sttistic ws used to test the ssumption of normlly distributed errors. If dt were not norml, nturl log trnsformtion of the dependent vrible or rnked dt ws used in the ANOVA. Homogeneity of vrince between tretments ws ssessed with Levene's test. If significnt differences in men vlues were indicted by the ANOVA F test, pired mens were evluted using the lest significnt difference (LSD) test. Dunnett's men seprtion test for unequl group vrinces ws used when heterogeneous group vrinces exceeded 3-fold difference between ny tretment pir (vn Belle 2002). The results from repeted groups were: 1) verged with the originl test group when compring tretment effects nd 2) compred to the initil test group for differences. The word repeted is used becuse the experiments did not occur t the sme time nd therefore re not replictes. Two sets of experiments were conducted, (1) to identify physiologicl nd swimming performnce indictors of cold shock, nd (2) indictors s functions of reduced rtion. The methods nd results of these two sets of tests re described here. Indictors of Cold Shock nd Effects on Swimming Performnce Methods A preliminry experiment incorporting declining tempertures ws conducted to determine the temperture of LOE for ech species. This informtion ws necessry to determine the tempertures to be used in cold shock tretments. I gve the shd (24 thredfin nd 18 gizzrd) n cute cold shock by decresing temperture 0.5 C/hr 13

23 beginning t the cclimtion temperture of 15 C. The temperture nd time since 15 C were recorded for ech individul when LOE occurred. The loss of swimming equilibrium ws used to determine LOE. Gizzrd shd did not lose equilibrium during testing t 3.4 C nd were removed becuse the chiller equipment ws not cpble of chilling the volume of wter ny colder. The tempertures used in the following cold shock experiments were bsed on the highest temperture (7.2 C for thredfin) ech species experienced LOE. Cold shock experimenttion begn with n cute cold shock (0.5 C/hr) to gizzrd shd terminting t test tempertures of 4 nd 5 C. I lso tested gizzrd shd tht were held for 6 hours t the 4 or 5 C test temperture to determine the effect of prolonged exposure t those tempertures fter temperture decline (Figure 9). The 5 C test group Temperture ( C) Gizzrd Shd Not Held Gizzrd shd Held 6 Hrs Thredfin Shd Not Held Thredfin Shd Held 3 or 6 Hrs Time (Hours) Figure 9. - Tretment grph displying the points (circles nd squres) where gizzrd nd thredfin shd were smpled during 0.5 C/hour cold shock experiments. Circles indicte groups smpled immeditely when the test temperture ws reched. Squres indicte groups smpled 3 or 6 hours fter reching the test temperture. 14

24 ws repeted 4 weeks fter the initil. Thredfin shd were given n cute cold shock to test tempertures of 7.5 nd 8.5 C. Thredfin shd were lso held for 6 hours t 8.5 C nd 3 hours t 7.5 C. I repeted the 8.5 C test group held for 6 hours three weeks fter the initil. Controls for both species were smpled t the cclimtion temperture of 15 C. The test groups were monitored for bnorml behvior or signs of distress during the cold exposure. The plsm cortisol (ng/ml) nd chloride (meq/liter) levels in ech pooled group were mesured to determine cute stress response to the test tempertures. Condition fctor ws clculted for ech individul to determine if significnt difference occurred between groups or if swimming performnce ws correlted with condition. Significnce ws tested t P=0.05 cross ll vribles nd test groups. Results The men LOE temperture for thredfin shd exposed to cold shock t 0.5 C/hr ws 5.8 C. LOE experienced by thredfin shd rnges from 7.2 C to 5 C. Only one thredfin shd hd not experienced LOE prior to 4.7 C t the time experiment ws terminted. No gizzrd shd experienced LOE prior to 3.4 C t the time experiment ws terminted (Figure 10). Using these preliminry results, the testing protocols were estblished for cold shock testing. Gizzrd Shd I observed signs of distress (bnorml response) in gizzrd shd during the cold shock tretments. Activity levels decresed s tempertures pproched 5 C nd fish becme totlly lethrgic by 4 C. There ws little response to vibrtion in the wter nd netting t 4 C. Gizzrd shd experiencing cold shock hd significntly lower men swimming times thn the control (P=0.0005) (Figure 11, Tble A1). Men swimming 15

25 Temperture ( C ) Thredfin Shd (LOE) Thredfin Shd (No LOE) Gizzrd Shd (No LOE) 18 Fish Time Since 15 C (Hours) Figure 10. Loss of equilibrium, s function of time nd temperture, for gizzrd nd thredfin shd subjected to cold shock tretment tht begn t 15 C nd declined t rte of 0.5 C/hour. The intended temperture decline is indicted by the line. Open dimonds re for shd tht lost equilibrium. Tringles re for shd tht were removed prior to LOE. Gizzrd Shd Men Swim Time (Min) b b c c 0 Control 5 C 5 C + 6Hrs 4 C 4 C + 6Hrs Figure Men (+1 SE) swimming time of gizzrd shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Groups were tested t 15 C (control), 5 C, fter 6 hours t 5 C (5C + 6Hrs), 4 C, nd fter 6 hours t 4 C (4 C + 6 hours). Tretments tht re sttisticlly different (P<0.05) hve different letters. 16

26 performnce time ws less in gizzrd shd cold shocked to 4 C thn t 5 C. Men swimming performnce of gizzrd shd held for n dditionl 6 hours t their test temperture ws not different sttisticlly from fish not held for n dditionl 6 hours. Men condition fctor ws not different between test groups or control (Figure 12). There ws no correltion between condition fctor nd men swimming performnce for gizzrd shd. The repeted group did not differ in swimming performnce compred to the initil group (Tble A3). Cold shock ffected cortisol nd chloride (P<0.0001) in gizzrd shd. Men plsm cortisol nd chloride were significntly higher in ll cold shocked groups compred to controls (Figure 13, Tble A1). Men plsm cortisol lso ws significntly less mong groups cold shocked t 5 C, 4 C, nd 4 C held for 3 hours. A significnt correltion ws found between swimming performnce nd cortisol (R 2 = ) nd chloride (R 2 = ) (Figure 14, 15). Men cortisol ws significntly lower in the repeted group compred to the initil group (Tble A3). Men plsm chloride did not differ mong cold shock tretments. The repeted group did not differ in men chloride compred to the initil group (Tble A3). Thredfin Shd I observed signs of distress in thredfin shd during the cold shock tretments t tempertures 2-3 C bove the LOE identified in the preliminry experiment. Individuls begn to swim out of sequence rther thn in school, often swimming into the side of the tnk s if serching for wrmer wter. The ctivity level of these fish ppered to 17

27 Gizzrd Shd Men Condition Fctor (x 1000) Control 5 C 5 C + 6Hrs 4 C 4 C + 6Hrs Figure Men (+1 SE) condition fctor of gizzrd shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Tretments tht re sttisticlly different (P<0.05) hve different letters. Gizzrd Shd Thredfin Shd Plsm Cortisol (ng/ml) b b c d b c b c Plsm Chloride (meq/l) b b Control 5 C 5 C + 6Hrs b b 4 C 4 C + 6Hrs c b b Control 8.5 C 8.5 C + 6Hrs c b 7.5 C 7.5 C + 3Hrs Figure Men (+1 SE) plsm cortisol nd chloride of gizzrd nd thredfin shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Tretments tht re sttisticlly different (P<0.05) hve different letters. 18

28 Men Swim Time (min) R 2 = Gizzrd Shd Thredfin Shd Liner (Gizzrd Shd) Liner (Thredfin Shd) R 2 = Men Cortisol (meq/l) Figure Liner correltion of men cortisol to men swim time of thredfin nd gizzrd shd. 70 Gizzrd Shd Thredfin Shd 60 Liner (Gizzrd Shd) Men Swim Time (min) R 2 = Liner (Thredfin Shd) R 2 = Men Chloride (ng/ml) Figure Liner correltion of men chloride to men swim time of thredfin nd gizzrd shd. 19

29 increse s tempertures decresed. There ws little response to vibrtion nd netting t 8.5 C. Cold shock hd significnt effect on swimming performnce of thredfin shd (P<0.01). Thredfin shd experiencing cold shock t declining tempertures to 7.5 C nd held for n dditionl 0 or 3 hours hd significntly shorter times for men swimming performnce thn controls nd the 8.5 C test group (Figure 16, Tble A1). The test group experiencing decline to 8.5 C + 6 hour holding ws different only from the 7.5 C + 3 hour group (Figure 16, Tble A1). Men condition fctor did not differ mong test groups, but two groups (the 8.5 C nd 8.5 C + 6 h) were less thn the control (Figure 17). There ws no correltion between condition fctor nd men swimming performnce for thredfin shd. The repeted group hd significntly longer men swimming performnce compred to the initil group (Tble A3). Men plsm cortisol ws significntly lower in ll thredfin shd test groups compred to the control (P<0.0001)(Figure 13, Tble A1). Test groups held for n dditionl 3 or 6 hours t their test temperture were significntly different from those smpled immeditely (Figure 13, Tble A1). No significnt correltion ws found between swimming performnce nd cortisol (R 2 = 0.283) nd chloride (R 2 = ) (Figure 14, 15). The repeted group hd significntly lower men cortisol compred to the initil group (Tble A3). Men plsm chloride ws significntly different in only the 8.5 C + 6 hour nd 7.5 C + 3 hour test group compred to controls (P=0.0034) (Figure 13, Tble A1). The 7.5 C test group hd significntly lower men chloride vlues compred to ll other test groups (Figure 13, Tble A1). The repeted group did not differ in men chloride compred to the initil group (Tble A3). 20

30 Thredfin Shd Men Swim Time (Min) b Control 8.5 C 8.5 C + 6Hrs 7.5 C 7.5 C + 3Hrs Figure Men (+1 SE) swimming time of thredfin shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Groups were tested t 15 C (control), 8.5 C, 8.5 C + 6 hours, 7.5 C, nd 7.5 C + 3 hours. Tretments tht re sttisticlly different (P<0.05) hve different letters. bc c Thredfin Shd Men Condition Fctor (x1000) b b b b Control 8.5 C 8.5 C + 6Hrs 7.5 C 7.5 C + 3Hrs Figure Men (+1 SE) condition fctor of thredfin shd exposed to cold shock tretment beginning t 15 C nd declining t rte of 0.5 C/hour to the test temperture. Tretments tht re sttisticlly different (P<0.05) hve different letters. 21

31 Indictors of Combined Coldshock nd Reduced Rtion nd Effects on Swimming Performnce Methods I fed ech test group reduced rtion of 0.5% of their mss in frozen brine shrimp for 14 nd 21 dys. The control groups were fed 5% of their mss in frozen brine shrimp for 14 dys. I then cold shocked gizzrd shd to temperture of 5 C nd thredfin shd t temperture of 8.5 C t rte of 0.5 C/hr. The 21-dy reduced rtion group ws repeted for ech species 1 dy fter the initil hd begun. The test groups were observed for chnges in swimming ctivity during the reduced rtion period. Fish were collected in Mrch of 2006 to determine if the level of rtion observed fter 21-dys ws similr to tht found in fish in lte winter. Following the reduced rtion period nd cold shock, blood ws withdrwn nd totl length (mm) nd weight (g) ws mesured for ech individul. I mesured plsm cortisol (ng/ml), chloride (meq/liter), totl protein (mg/dl), triglycerides (mg/dl), nd hemtocrit (%) level in ech pooled group. Totl protein nd triglycerides were determined using centrificl fst nlyzer (Cobs brnd). The Cobs is n utomted spectrophotometer tht opertes s centrificl fst nlyzer for testing tht cn be progrmmed to test multiple smples t once. Results Gizzrd Shd Gizzrd shd remined ctive during the reduced rtion periods. Groups fed reduced rtion did not hve significntly different men swimming performnce thn 22

32 controls (P=0.6943) (Figure 18, Tble A2). A tretment effect of reduced rtion ws observed in both cortisol nd chloride (P<0.0001). The men cortisol level of the 14-dy reduced rtion group ws higher compred to the 21-dy reduced rtion group nd control (Figure 19, Tble A2). The men plsm chloride levels of both reduced rtion groups ws different from controls. The 14-dy nd 21-dy rtion groups lso differed in men plsm chloride. Tretment effects were lso observed in totl protein (P<0.01), triglycerides (P<0.0001), hemtocrit (P<0.01), nd condition fctor (P<0.01). Men plsm totl protein ws significntly higher in the 21-dy reduced-rtion group thn the 14-dy reduced-rtion group (Figure 20, Tble A1). Men plsm triglycerides decresed between 14 nd 21 dys of reduced rtion. Men hemtocrit nd condition fctor ws lower only in the 21-dy test group compred to the control; the 14-dy reduced rtion group did not differ from the controls for either. Men condition fctor ws lower in the 21-dy group nd control. Gizzrd shd collected in Mrch of 2006 hd lower men condition fctors (K=7.4) compred to the condition resulting from 21-dys of reduced-rtion tretment in this study (K=8.1). Men swimming performnce nd men cortisol were significntly lower in the repeted 21-dy reduced rtion group compred to the initil 21-dy reduced rtion group (Tble A3). No other differences were found between the repeted nd initil group. Thredfin Shd Thredfin shd schooled nd remined ctive during the reduced-rtion test period. Groups fed reduced rtion did not hve significntly different men swimming performnce compred to controls (P= ) (Figure 18, Tble A2). Tretment effects were not found in cortisol (P=0.5975) or chloride (P= )(Figure 19, Tble A2). 23

33 Gizzrd Shd Men Swim Time (Min) Thredfin Shd Men Swim Time (Min) Control 14 Dys 21 Dys Figure Men (+1 SE) swimming time of gizzrd nd thredfin shd exposed to cold shock fter one of three protocols:14 dys of full rtion, 14 dys of reduced rtion, or 21 dys of reduced rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters. Plsm Cortisol (ng/ml) Gizzrd Shd b Thredfin Shd Plsm Chloride (meq/l) b Control 14 Dys c 21 Dys Control 14 Dys 21 Dys Figure Men (+1 SE) plsm cortisol nd chloride of gizzrd nd thredfin shd exposed to cold shock fter one of three rtion tretments:14 dys of full rtion, 14 dys of reduced rtion, or 21 dys of reduced rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters. 24

34 Plsm Totl Protein (mg/dl) Gizzrd Shd b b Thredfin Shd b b Plsm Triglyceides (mg/dl) b c b Hemtocrit (%) b b c Condition Fctor (x 1000) Control 14 Dys b 21 Dys Control 14 Dys b 21 Dys Figure Men (+1 SE) condition fctor, hemtocrit, plsm totl protein, nd plsm triglycerides of gizzrd nd thredfin shd exposed to cold shock fter one of three rtion tretments:14 dys of full rtion, 14 dys of reduced rtion, or 21 dys of reduced rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters. 25

35 Tretment effects were observed in totl protein (P=0.0001), triglycerides (P<0.0001), hemtocrit (P<0.0001), nd condition fctor (P<0.01). Men plsm totl protein ws significntly lower in the reduced rtion groups thn in the control, but there ws no difference between the two reduced rtion groups. Men plsm triglycerides were significntly higher in the14-dy reduced-rtion group thn controls nd 21-dy reducedrtion group. Hemtocrit ws significntly lower in both reduced rtion groups thn the control nd men hemtocrit ws lower for the 21-dy reduced- rtion group thn the 14- dy reduced-rtion group. Men condition fctor ws significntly lower in the 21-dy reduced-rtion group compred to 14-dy reduced-rtion group nd control which were not different (Figure 20, Tble A1). Higher men cortisol nd lower men triglycerides were the only differences between the repeted nd initil 21-dy reduced rtion groups. Gizzrd shd collected in Mrch of 2006 hd lower men condition fctors (K=6.7) compred to the condition resulting from 21-dys of reduced-rtion tretment in this study (K=6.9). 26

36 III. Discussion The preliminry experiment yielded LOE tempertures within the reported rnge of previous studies for both species. None of the 18 gizzrd shd lost equilibrium by the time 3.4 C ws reched. The experiment ws ended t this point due to time nd equipment limittions. Since the initil LOE temperture for gizzrd shd ws not determined, we could not estblish equivlent degrees bove LOE for our further testing of both species. More testing is needed to identify the point of LOE of gizzrd shd but the results for both species show similr chnges in physiologicl nd behviorl indictors t the tempertures I tested. Signs of behviorl distress during cold shock prior to LOE or deth in thredfin shd hve been reported by others. Thredfin shd exposed to cute temperture declines begn showing signs of behviorl distress s much s 5 C prior to mortlity nd lck of response to movement nd vibrtion 6-7 C bove lethl tempertures (Griffith 1978). Moribund thredfin shd, exposed to 1-4 C temperture declines in 4 hours, swm individully rther thn in schools prior to LOE (Griffith nd Tomljnovich 1975). Studies reporting gizzrd shd showing signs of distress or response prior to LOE were not found. The swimming performnce, cortisol, nd chloride levels of gizzrd shd chnged in reltion to cold shock tretment. I found tht wter tempertures of 5 C nd less ffect the swimming performnce of gizzrd shd nd render this species more susceptible to impingement. Holding gizzrd shd for n dditionl six hours t test temperture did not result in significntly lower men swimming performnce, but response of 27

37 reduced performnce with incresed exposure is strongly suggested. Condition fctor did not differ between ny given groups of gizzrd shd used in the swimming performnce test. This is importnt becuse I collected gizzrd shd over three month period during which environmentl fctors could hve influenced condition. Cortisol nd chloride were positively correlted with swimming performnce nd could be potentil indictors of susceptibility to impingement. Cortisol ws expected to increse s response to cold shock induced stress, which hs been observed in similr studies. Hyvrinen et l. (2004) found incresing cortisol levels when brown trout were cold shocked in n ice bth. In most species, chloride chnges re the inverse of cortisol but in this cse chloride response ws similr to cortisol nd most likely the correltion found is not vlid. Dvis (2004) lso documented the trend of incresing cortisol nd decresing chloride s response to stressor (cute confinement). All test groups of gizzrd shd experienced increses in both cortisol nd chloride. The cortisol level in the control group ws higher thn resting in most fish species (0-50 ng/ml), however no published reserch involving shd cortisol levels ws found for comprison. The chloride vlues were lower in ll groups of gizzrd shd thn the published rnge ( meq/liter) for unstressed fish (Brton et l. 2002). The cclimtion period of one week to 15 C my not hve been sufficient to stbilize osmoregultory function in this species. The swimming performnce, cortisol, nd chloride levels of thredfin shd responded to the cold shock tretment. The swimming performnce of thredfin shd ws similr to tht of gizzrd shd when exposed to cold shock tretments. Wter tempertures of 7.5 C nd less reduced the swimming performnce of thredfin shd nd possibly renders this species more susceptible to impingement thn gizzrd shd. 28

38 Similrly, study by Griffith nd Tomljnovich (1975) showed the bility of thredfin shd to resist impingement ws severely impired t tempertures below 8 C, but t higher tempertures impingement ws slightly or not t ll impired. One individul in the control group did not swim the 60 minute mximum, which cused the 8.5 C test groups mens to not differ from the control. If fish were llowed to swim greter thn 60 minutes, the 8.5 C men probbly would hve differed from the control. Holding thredfin shd for n dditionl 3 or 6 hours t their test temperture did not result in significntly lower men swimming performnce, but response of reduced performnce with incresed exposure is strongly suggested. Condition fctor differed between severl groups of thredfin shd used in the swimming performnce tests. However, condition fctor ws not correlted with men swim time. Cortisol nd chloride were not correlted with swimming performnce. Cortisol ws expected to increse s response to cold shock induced stress, but I found the opposite response. The unexpected cortisol chnges could be due to: 1) reduced bility of shd to mount stress response under bnormlly cold tempertures or 2) the control group experienced unknown stressors such s reduced wter qulity or effects due to hndling stress tht resulted in higher cortisol levels. Strnge (1980) documented the reduced bility of chnnel ctfish to mount stress response t cold tempertures. Dvis (2004) lso showed tht fish held t colder tempertures hd delyed responses in cortisol nd chloride in comprison to those fish held t wrmer tempertures. However, it is most likely tht the control group ws stressed for n unknown reson. If the cortisol levels were in the norml resting rnge of most fish species the expected chnges (increse in response to stressor nd decrese during cclimtion) in cortisol would be present. The groups held for n dditionl 3 or 6 29

39 hours showed decreses in cortisol compred to those not held which could represent recovery or cclimtion to the cold tempertures. Strnge (1980) showed tht chnnel ctfish becme cclimted to stress fter severl dys nd cortisol declined. Similrly, Strnge nd Schreck (1978) showed tht cortisol levels begn to decrese 3.5 hours fter stressor ws presented to juvenile Chinook slmon. The expected chloride reltionship, n inverse of the cortisol response, ws found in thredfin shd. All test groups experienced increses in chloride when cortisol decresed nd vice vers. Similr to gizzrd shd, the chloride vlues re lower in ll groups thn the published rnge ( meq/liter) for unstressed fish (Brton et l. 2002). The cclimtion period of one week t 15 C my not hve been sufficient to stbilize osmoregultory function in this species. The repeted cold shock test groups showed tht cortisol levels were ~ 20 ng/ml lower in both species compred to the initil groups. The lower men cortisol observed in repeted cold shock group of gizzrd shd pprently did not ffect their swimming performnce becuse levels were still bove norml resting vlues. However, the men swimming performnce of the repeted cold shock group of thredfin shd ws ffected by the decrese in cortisol nd is possibly relted to the lower cortisol vlue being similr to resting levels. Johnsen et l. (1994) found tht when cortisol levels of rinbow trout incresed bove resting levels due to stressor (toxicnt), swimming performnce decresed. The drop in cortisol in both species could be relted to the difference in time tht tests were performed. The initil test groups were performed on shd tht were collected t C, wheres the repeted groups were collected when wter tempertures were closer to 20 C. The experimentl methods included one week 30

40 cclimtion period to mbient lbortory temperture of bout 22 C followed by week t 15 C, but this my not hve been long enough to offset the difference in field cclimtion temperture. This supports the need for more reserch involving cclimtion temperture. Previous studies on vriety of fish species indicted tht swimming performnce, totl protein, triglycerides, hemtocrit, nd condition fctor would decline s the durtion of the reduced rtion incresed. Mrtinez et l. (2004) demonstrted tht Atlntic cod (Gdus morhu) tht were strved hd reduced swimming performnce compred to cod tht were fed. McMilln nd Houlihn (1991) reported rinbow trout hving reduced serum totl protein fter severl dys of fsting. Rune et l. (2002) compred triglyceride levels in common crp (Cyprinus crpio) fed high rtion to those fed low rtion nd found tht triglyceride levels were lower in those fed low rtion. Adms et l. (1985) reported lower hemtocrit nd condition fctor levels in stressed gizzrd shd compred to unstressed shd. Gizzrd nd thredfin shd showed little response in men swim time or physiologicl indictors fter 14 nd 21 dys of reduced rtion when subjected to cold shock. It is possible tht stress response occurred nd the shd dpted to the lck of feeding or tht the period of strvtion ws not long enough to illicit n ffect. Since these species typiclly experience periods of low food vilbility in winter, possible dpttion to periods of reduced feeding could occur, therefore helping to explin, in prt, the lck of cler response in the lb to reduced rtion. The repeted group (21-dy reduced rtion) for gizzrd shd showed decresed cortisol levels nd decrese in swimming performnce. However, the cortisol levels were still bove norml resting vlues. The repeted group (21-dy reduced rtion) for 31

41 thredfin shd differed only in cortisol (which is probbly not biologiclly significnt with men cortisol differences of only 6 ng/ml) nd triglycerides. To better understnd the implictions of reduced rtion, I collected gizzrd nd thredfin shd from the Clinch River in Mrch These fish hd significntly lower condition fctors, (7.4 for gizzrd nd 6.7 for thredfin shd) thn those held for 21 dys under reduced rtion (8.1 for gizzrd nd 6.9 for thredfin shd). Therefore, even though the reduced rtion period of 21 dys did result in poorer condition of fish compred to controls, condition of fish in the lb did not pproximte tht of shd collected from the reservoir in lte winter. If the condition of fish in the lb hd been similr to tht of shd collected in the field in lte winter, the expected declines in nutritionl sttus indictors nd swimming performnce my hve occurred in those fish subjected to the cold shock tretment. Chnges in hemtocrit nd condition fctor were significnt in thredfin shd fter 21-dys of reduced rtion. This differentil response between species is likely due to gizzrd shd storing proportiontely greter mounts of lipids thn thredfin shd (Adms 1999). Hemtocrit nd condition fctor re reltively simple indictors tht could be used in the field to rpidly determine susceptibility of shd to impingement. These re the types of rpid ssessment indictors tht would be necessry to demonstrte fish were impired prior to impingement in order to meet EPA protocol requirements. However, the pplicbility of these mesures nd other esily nd rpidly pplied indictors of susceptibility should be further ssessed by investigting physiology nd swimming performnce of shd whose condition simultes the condition of shd collected from the reservoir in lte winter. 32

42 Severl hybrid shd were found during the study. They closely resembled thredfin shd with the exception of blunt snout overhnging the lower jw tht is typicl of gizzrd shd (Figures A4, A5). This is not uncommon: Minckley nd Krumholz (1960) nd Shelton nd Grinsted (1973) documented cses of hybridiztion between these species. Dt collected from these fish were not used in the nlyses. Testing hybrid shd my be needed to determine if they respond differently to cold shock nd reduced rtion. Prcticl Implictions nd Future Studies The overll results of this study indicte tht cold tempertures, prticulrly those bove the tempertures of LOE, render gizzrd nd thredfin shd more susceptible to impingement. Potentil indictors of susceptibility to impingement hve been identified in this study (e.g., hemtocrit nd condition fctor) nd could be performed on fish deemed susceptible to impingement in the vicinity of intke structures. The use of multiple indictors of stress ids in ccounting for or helping to explin confounding stressors which my be present in nturl ecosystems. This study hs shown tht gizzrd nd thredfin shd susceptible to impingement could be clssified s either impired or moribund. It is ssumed tht moribund fish would not recover nd die regrdless of impingement. Impired shd could hve recovered if environmentl conditions improved nd therefore would not hve died if not impinged. However, impired shd re more susceptible thn helthy shd to nturl predtion. In comprison to nturl predtion, power plnts could be considered selective predtors tht re removing the wek individuls from the popultion tht would likely hve been predted. Severl studies hve relted cold shock to incresed predtion rtes 33

43 in fish (Coutnt et l. 1974; Coutnt et l. 1976; Wolters nd Coutnt 1976). The current USEPA regultions stte tht ccounting for moribund fish cn be used to meet impingement reductions of 80-95% t existing intkes. The USEPA could consider revising the moribund clssifiction of fish to include environmentlly impired shd s well, bsed on tempertures few degrees bove the species LOE tempertures. However, chnging the clssifiction of fish tht cn be used to meet impingement reductions must be supported with strong scientific evidence of the predtion reltionship. The issue of impired fish versus moribund fish would be more significnt t existing intkes locted in wrmer climte res where moribundity is less likely. Using physiologicl nd performnce-level indictors to ssess impingement susceptibility ppers promising, but further studies re necessry to evlute the reltive importnce of cold shock regimes nd nutritionl sttus to impingement susceptibility. More testing is needed for both species on the effects of: 1) rte of temperture decline reltive to the cclimtion temperture, 2) lower cclimtion tempertures reltive to the cold shock test tempertures, nd 3) longer cclimtion period prior to testing. Further reserch ddressing the role nd importnce of nutritionl sttus should include: 1) longer reduced rtion tretments using fish collected in lte summer, 2) combining severl different cold shock tempertures with the reduced rtion tretment, 3) the recovery of fish held under reduced rtion nd cold shocked. This type of informtion would clrify the reltionship of the physiologicl indictors to the susceptibility of impingement of shd. 34

44 List of References 35

45 Adms, S.M., J.E. Breck, nd R.B. McLen Cumultive stress-induced mortlity of gizzrd shd in southestern U.S. reservoir. Environmentl Biology of Fish 13: Adms, S.M., W.D. Crumby, M.S. Greeley, Jr., M.G. Ryon nd E.M. Schilling Reltionships between physiologicl nd fish popultion responses in contminted strem. Environmentl Toxicology nd Chemistry 11: Adms, S.M Ecologicl role of lipids in the helth nd success of fish popultions. Pges in M.T. Arts nd B.C. Winmn, editors. Lipids in freshwter ecosystems. Springer-Verlg NY, NY. Brton, B.A, J.D. Morgn, nd M.M. Vijyn Physiologicl nd condition-relted indictors of environmentl stress in fish. Pges in S.M. Adms, editor. Biologicl indictors of qutic ecosystem stress. Americn Fisheries Society, Bethesd, Mrylnd. Bodol, A Life history of the gizzrd shd, Dorosom cepedinum (LeSeuer), in Western Lke Erie. U.S. Fish nd Wildlife Service Bulletin 65(2):391. Coutnt, C.C., H.M. Duchrme Jr., nd J.R. Fischer Effects of cold shock on vulnerbility of juvenile chnnel ctfish (Ictlurus puncttus) nd lrgemouth bss (Micropterus slmoides) to predtion. Journl of Fisheries Resources Bord Cnd 31: Coutnt, C.C., D.K. Cox, nd K.W. Moored Jr Further studies of cold shock effects on susceptibility of young chnnel ctfish to predtion. Therml Ecology II, ERDA Symposium, Svnnh River Ecology Lbortory, Georgi, p Cox, D.K. nd C.C. Coutnt Acute cold-shock resistnce of gizzrd shd. Therml Ecology II, ERDA Symposium, August, Georgi, p.159. Dvis, K.B Temperture ffects physiologicl stress responses to cute confinement in sunshine bss (Morone chrysops x Morone sxtilis). Comprtive Biochemistry nd Physiology Prt A: Dutil, J., Y. Lmbert, nd D. Chbot. Winter nd spring chnges in condition fctor nd energy reserves of wild cod compred with chnges observed during fooddeprivtion in the lbortory. ICES Journl of Mrine Science 60.4 (2003): EPRI Lrge-Scle Nturl Mortlity Events in Clupeid Fishes: A Literture Review,, Plo Alto, CA. EPRI Report. 36

46 Griffith, J. S. nd D. A. Tomljnovich Susceptibility of thredfin shd to impingement. Proceedings of the 29th Annul Conference of the Southestern Assocition of Gme nd Fish Commissioners Griffith, J. S Effects of low temperture on the survivl nd behvior of thredfin shd, Dorosom petenense. Trnsctions of the Americn Fisheries Society. 107(1): Heidinger, R.C Life history of the gizzrd shd nd thredfin shd s it reltes to the ecology of smll lke fisheries. Proceedings of Smll Lkes Mngement Workshop- Pros nd Cons of Shd. Iow Conservtion Commission nd Sport Fishery Institute, Des Moines. Hyvrinen, P., S. Heinim, H. Rit Effects of brupt cold shock on stress responses nd recovery in brown trout exhusted by swimming. Journl of Fish Biology 64: Johnsen, J. A., C.J. Kennedy, R.M. Sweeting, A.P. Frrell, nd B.A. McKeown Sublethl Effects of Tetrchloroguicol on Juvenile Rinbow Trout, Oncorhynchus Mykiss, Following Acute nd Chronic Exposure. Cndin journl of fisheries nd qutic sciences 51.9: Lor, J. M., J. S. Griffith, nd K. D. Kumr An nlysis of fctors influencing the impingement of thredfin shd t power plnts in the Southestern United Sttes. Fourth Ntionl Workshop on Entrinment nd Impingement Mrtinez, M., M. Bedrd, J.D. Dutil, nd H. Guderley Does condition of Atlntic cod (Gdus morhu) hve greter impct upon swimming performnce t U crit or sprint speeds? The Journl of Experimentl Biology 207: McLen, R. B., P. T. Singley, J. S. Griffith, nd M. V. McGee Thredfin shd impingement: effect of cold stress. Report to the Nucler Regultory Commission. Publiction No McLen, R. B., J.J. Beuchmp, V.E. Kne, nd P. T. Singley Impingement of thredfin shd: effects of temperture nd hydrogrphy. Environmentl Mngement 6 (5): McLen, R. B., J. S. Griffith, nd M. V. McGee Thredfin shd, Dorosom petenense Günther, mortlity: cuses nd ecologicl implictions in Southestern United Sttes reservoir. Journl of Fish Biology. 27:1-12. McMilln, D.N., nd D.F. Houlihn Protein synthesis in trout liver is stimulted by both feeding nd fsting. Fish Physiology nd Biochemistry 10(1):

47 Minckley, W.L., nd L.A. Krumholz Nturl hybridiztion between the clupeid gener Dorosom nd Signlos, with report on the distribution of S. petenensis. Zoologic. 44(4): NtureServe NtureServe Explorer: An online encyclopedi of life [web ppliction]. Version 4.6. NtureServe, Arlington, Virgini. Avilble: (Accessed: Februry 26, 2006). Neumnn, Dvid A., W.J. Wchter, E.L. Melinsky nd D.G. Brdrik Field nd lbortory ssessment of fctors ffecting the occurrence nd distribution of gizzrd shd (Dorosom cepedinum) t Front Street Stem Electric Genertion Sttion, Erie, Pennsylvni. Pennsylvni Electric Compny. Rune, N.M., E.A. Huismn, nd J. Komen The influence of feeding history on the cute stress response of common crp. Aquculture 210(1-4): Strnge, R. J., nd C. B. Schreck Cortisol concentrtions in confined juvenile Chinook slmon (Oncorhynchus Tshwytsch ). Trnsctions of the Americn Fisheries Society 107(6): Strnge, R. J Acclimtion temperture influences cortisol nd glucose concentrtions in stressed chnnel ctfish. Trnsctions of the Americn Fisheries Society 109: Strwn, K Resistnce of thredfin shd to low tempertures. Proceedings of the Annul Conference of the Southestern Assocition Gme nd Fish Commission. 17: U.S. Army Corps of Engineers TN river nvigtion chrts. U.S. Army Corps of Engineers. Avilble: (Accessed: Februry 26, 2006). Vn Belle, Gerld Sttisticl rules of thumb. Pge 10 in Wiley series in probbility nd sttistics. John Wiley & Sons, NY, NY. White, A.M., F.D. Moore, N.A. Alldridge, nd D.M. Loucks The effects of nturl winter stresses on the mortlity of the estern gizzrd shd, Dorosom cepedinum, in Lke Erie. Environmentl Resource Assocites, Inc. Report 78. Clevelnd, Ohio. Wolters, W.R. nd Coutnt, C.C., The effects of cold shock on the vulnerbility of young bluegill to predtion. Therml Ecology II, ERDA Symposium, Svnnh River Ecology Lbortory, Georgi, p

48 Appendix 39

49 Tble A1. - Men (± SE) swimming performnce, plsm cortisol, nd plsm chloride of gizzrd nd thredfin subjected to cold shock. Groups of 34 gizzrd shd were cold shocked t 0.5 C per hour to one of the following smpling tempertures: 5 C nd held for 0 hours, 5 C nd held for 6 hours, 4 C nd held for 0 hours, or 4 C nd held for 6 hours. Groups of 24 thredfin shd were cold shocked t 0.5 C per hour to one of the following smpling tempertures: 8.5 C nd held for 0 hours, 8.5 C nd held for 6 hours, 7.5 C nd held for 0 hours, or 7.5 C nd held for 3 hours. Controls for both species were smpled t their cclimtion temperture of 15 C. Tretments tht re sttisticlly different (P<0.05) hve different letters. Swimming Plsm Plsm Tretment Performnce cortisol chloride Group (minutes) (ng/ml) (meq/liter) Gizzrd Shd Control 60.0 ± ± ± C + 0Hrs 43.0 ± 5.4 b 79.1 ± 2.5 b 94.9 ± 0.6 b 5 C + 6Hrs 36.6 ± 8.9 b 77.4 ± 4.8 b 94.4 ± 0.3 b 4 C + 0Hrs 17.0 ± 7.5 c ± 1.9 c 95.6 ± 0.6 b 4 C + 6Hrs 4.9 ± 1.1 c 89.7 ± 4.8 d 95.8 ± 0.5 b Thredfin Shd Control 54.9 ± ± ± 1.1 c 8.5 C + 0Hrs 47.1 ± ± 2.6 b 93.0 ± 0.8 b 8.5 C + 6Hrs 38.4 ± 6.1 b 32.1 ± 2.4 c 94.3 ± 0.4 b 7.5 C + 0Hrs 18.2 ±10.8 bc 62.0 ± 6.6 b 90.5 ± 1.4 c 7.5 C + 3Hrs 16.4 ± 6.5 c 32.9 ± 2.5 c 93.8 ± 1.0 b 40

50 Tble A2. - Men (SE) condition fctor, plsm cortisol, chloride, totl protein, nd triglycerides of gizzrd nd thredfin shd fed reduced rtions nd cold shocked. Groups of 18 gizzrd shd or 24 thredfin shd were fed 0.5% body weight rtion per dy for 14 or 21 dys. Gizzrd shd were then cold shocked t 0.5 C per hour to 5 C nd held for 0 hours. Thredfin shd were then cold shocked t 0.5 C per hour to 8.5 C nd held for 0 hours. Controls for both species were fed full rtion. Tretments tht re sttisticlly different (P<0.05) hve different letters. Tretment Group Swimming Performnce (minutes) Plsm cortisol (ng/ml) Plsm chloride (meq/liter) Plsm Totl Protein (mg/dl) Plsm Triglycerides (mg/dl) Hemtocrit (%) Condition Fctor Gizzrd Shd Control 50.3 ± ± ± ± 1.9 b 69.6 ± ± ± Dys 42.5 ± ± 5.6 b 93.5 ± 0.8 b 90.0 ± ± 3.2 b 22.0 ± ± Dys 48.9 ± ± ± 0.7 c 96.7 ± 1.1 b 82.4 ± 1.8 c 21.2 ± 0.2 b 8.1 ± 0.05 b Thredfin Shd Control 59.2 ± ± ± ± ± ± ± Dys 51.0 ± ± ± ± 1.7 b 58.3 ± 5.0 b 17.5 ± 0.4 b 7.0 ± Dys 53.3 ± ± ± ± 0.9 b 36.0 ± ± 0.2 c 6.9 ± 0.03 b 41

51 Tble A3. A comprison of dependent vrible mens of originl nd repeted test groups. The 4 C test group (cold shock) nd the 21 dy test group (reduced rtion nd cold shock) were repeted in gizzrd shd. The 8.5 C + 6 hrs test group (cold shock) nd the 21 dy test group (reduced rtion nd cold shock) were repeted in thredfin shd. Significnt differences (P<0.05) in mens re indicted by sterisks with the p-vlue in prentheses. Dependent Vribles (test group repeted) Originl Tretment Repet Tretment Significnce (P< 0.05) Cold Shock Gizzrd Shd (4 C test group) Swim Time (0.9382) Cortisol *(< ) Chloride (0.0871) Thredfin Shd (8.5 C + 6 hrs test group) Swim Time *(0.0049) Cortisol *(0.0001) Chloride (0.9568) Reduced Rtion + Cold Shock Gizzrd Shd (21 dy test group) Swim Time *(0.0250) Cortisol *(< ) Chloride (0.8498) Totl Protein (0.4605) Triglycerides (0.5877) Hemtocrit (0.3960) Condition Fctor (0.8743) Thredfin Shd (21 dy test group) Swim Time (0.3722) Cortisol *(0.0400) Chloride (0.1453) Totl Protein (0.1670) Triglycerides *(0.0008) Hemtocrit (0.2891) Condition Fctor (0.1248) 42

52 Figure A4. - Two hybrid shd t top of picture followed by three thredfin shd nd single gizzrd shd. Notice the similrity in fin colortion but different snout shpe. The hybrid lso hd the long dorsl fin filment (not visible in picture) chrcteristic of thredfin shd. 43

53 Figure A5. Gizzrd shd (top) compred to two hybrid shd (middle nd bottom). Notice the yellow fin colortion, rounded overhnging snout, nd long dorsl filment. 44